Devices and methods for isolating and recovering target cells

ABSTRACT

A cell isolating device and method is provided to concentrate or isolate cells with specific characteristics from a mixture of different cell types. One embodiment may comprise two subtypes of antibodies that are directly conjugated to biotin (Ab b ) and conjugated to a fluorescent molecule (Ab f ). The conjugated antibodies (Ab b +Ab f ) bind to the target cells in a mixed cell suspension. The cell suspension is then passed over an immobilized avidin or streptavidin substrate on a glass microscope slide. The biotinylated target cells adhere to the avidin/streptavidin substrate, while the unbound cells are washed off and collected in a wicking member. Captured cells on the avidin/streptavidin substrate may then be visualized directly using a fluorescent microscope or detected and enumerated via an on-board fluorescent detection device. Additional chemicals and/or physical manipulation may then be applied to the device to release viable target cells for subsequent analysis.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/364,679, filed Mar. 15, 2002, and whose entirecontents are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] All living bodies are comprised of individual cells, each celldefining an environment where various biological and chemical reactionstake place. In particular, each cell contains a cell membrane thatseparates the internal environment of the cell from the externalenvironment and thereby controls the entry and exit of various nutrientsand waste. Additionally, the cell membrane includes various proteins,sugars, and other molecules that “identify” a particular cell type,these identifying molecules commonly being referred to as antigens.

[0003] In order to better understand the function and pathologies ofcells, numerous methods have been developed to isolate and concentrate adesired target cell population from a mixed cell population so that thetarget cell population can be further analyzed. One such method is basedupon cell density wherein a mixture of cells is spun at high speeds in acentrifuge so that the higher density cells become separated for thelesser density cells. Although this method is effective at separatingdifferent cells, centrifugation does not have good cell-separationspecificity as different types of cells may have the same or similarcell density.

[0004] Accordingly, more sophisticated cell separation techniques havebeen developed wherein cells are separated based upon the presence ofcertain cellular identifiers, namely, antigens, found on the cellularmembrane. More specifically, these selection methods are based uponusing antibodies that react with antigens found on a particular targetcell membrane. In one such method, the antibodies are affixed on thesurface of a substrate, such as magnetic beads or small iron-coatedparticles. When mixed with the cell sample, the antibody-coated beads orparticles bind to the specific antigens on the cell membrane. As asample cell solution is passed through a magnetic separation column, themagnetic particles with the target cells attached then bind to thesurface of the magnetic field. The target cells are then released fromthe column by removing the magnetic field from the cell separationcolumn. Other known methods use variations of target cell binding incontinuous-flow “immunoaffinity” columns. Generally, with immunoaffinitycolumns, once the target cells bind to the column by antigen-antibodyaffinity, the bound target cells are released by mechanically agitatingthe immunoaffinity column.

[0005] Cell separation techniques based upon cellular membraneidentifiers are particularly useful in isolating specific cells as suchtechniques may be modified or tailored for specific target cells.Indeed, such highly specific cell separation techniques are particularlyuseful for diagnosing and treating specific and potent diseases such as,but not limited to, autoimmune diseases or cancer.

[0006] The utility of immunoseparation techniques as a diagnostic toolis evident given the prevalence of various diseases. Cancer, forinstance, is expected to afflict approximately 1.3 million people in2002 and result in approximately 500,000 deaths. Studies have shown,however, that early detection of cancer results in improved survivalrates as treatment is more likely to be successful during the earlystages of cancer. Yet while early diagnosis and treatment increases thechances of survival, there still remains the possibility of relapse.Accordingly, there has been considerable research into the causes ofcancer relapse.

[0007] In particular, over the past 12 years, numerous research studieshave been designed to track the presence of low numbers of micrometastictumor cells (so called “micrometastases”) in blood, bone marrow, andeffusion fluids in patients with cancer. Studies have shown that thepresence of tumor micrometastases in blood and bone marrow at time ofsurgery is a strong prognostic indicator of poor prognosis and earlyrelapse in breast, prostate, ovarian, and lung cancer patients.Furthermore, the reappearance of circulating tumor cells followingchemotherapy appears to herald the earliest indication of diseaserecurrence. Accordingly, the early detection of these micrometastasesmay result in higher survival rates for patients in relapse.

[0008] While the presence of micrometastases are strong indicators ofcancer, these tumor cells are particularly difficult to detect as thereported frequency of micrometastatic tumor cells range from 1-5micrometastatic tumor cells per 100,000-1,000,000 bone marrow cells andfrom 1 micrometastatic tumor cell in 1,000,000 to 100,000,000 bloodcells. Despite the low frequency of micrometastases, various methodshave been developed to concentrate or isolate the micrometastatic cellsfrom blood, bone marrow, or effusion fluids using immunoselectionmethods such as, but not limited to, immunomagneticseparation/isolation, immunocolloidal separation/isolation, or flowcytometric separation/isolation.

[0009] While these prior art immunoselection methods have proven useful,these methods can be inefficient as they require considerable operatorintervention during the separation process. For instance, the separationcolumn usually needs cleaning and priming prior to the introduction of asample solution. Furthermore, the column requires constant monitoringduring the separation process. As a result, the efficiency, accuracy,and recovery of targeted cell is often directly related to operatorskill or error. Accordingly, it is desirable to have a cell separationdevice that minimizes operator error.

[0010] Moreover, the design of prior art immunoseparation columns mayalso hinder the recovery of a targeted cell. For instance,immunomagnetic separation/isolation methods result in permanent orsemi-permanent adherence of magnetic beads/particles to the isolatedcells. Accordingly, the difficulty and sometimes inability to remove thetarget cells from the magnetic beads reduces the accuracy of thesemethods. For example, isolated target cells may become damaged when thecells are separated from the column as relatively harsh chemical ormechanical processes are typically required to remove the target cellsfrom the beads. This is particularly problematic when attempting todetect cells, such as micrometastases, which have a low frequency.

[0011] Furthermore, target cell recovery is predicated on having theproper target cell to magnetic bead ratio. If the target cell to beadratio is not properly optimized, “background” interference may developdue to the presence of beads or particles that are not bound to thetarget cells thereby reducing the method's accuracy. However, optimizingthe target cell to bead ratio is difficult as the frequency of thetarget cell is usually unknown.

[0012] Accordingly, there remains a need for devices and methods thatoptimize target cell isolation, purity, and viability. There alsoremains a need for devices and methods that isolate viable,uncompromised cells (physically and/or biochemically) so as to enablesubsequent analysis and potential therapeutic applications of theisolated cells.

BRIEF SUMMARY OF THE INVENTION

[0013] The present invention relates to a device and method ofconcentrating or isolating a target cell from a mixture having differentcell types. More specifically, the target cells are isolated onto afixed substrate through the use of a bifunctional molecule wherein afirst functional group of the molecule is reactive with the fixedsubstrate and the second functional group is reactive with the targetcell. That is, target cells may be more easily recovered and identifiedbecause the steps of isolation and identification are carried out on thesame substrate. In contrast, prior art devices and methods typicallyrequire two or more substrates to isolate and identify the target cell.In an exemplary embodiment, the present invention may be utilized in thedetection of rare cellular events (e.g., tumor cells in blood, bonemarrow, effusion fluids, virally infected cells, cells carrying aberrantgenetic information).

[0014] According to an exemplary embodiment, the cell separating devicecomprises a substantially planar surface having a bioactive coatingapplied thereon and at least one bifunctional compound capable ofbinding to said target cell and to said bioactive coating. Thebifunctional compound allows for the isolation of the target cell from acellular mixture.

[0015] According to another exemplary embodiment, the cell separatingdevice comprises a substantially planar surface having a bioactivecoating applied thereon. The device further includes a conduit in spacedrelationship with the planar surface, wherein the conduit includes atleast one channel to deliver a cellular mixture to the bioactivecoating. The device also includes a fluid absorbing media provided onthe planar surface and positioned adjacent to the bioactive coating.

[0016] The present invention also provides methods of isolating a targetcell from a cellular mixture. According to the teachings of the presentinvention, the method comprises the steps of providing a cell separationdevice having a planar surface coated with a bioactive coating and abifunctional compound. The bifunctional compound is combined with thecellular mixture, and this mixture is then exposed to the bioactivecoating. The bioactive coating is then analyzed for the presence of thetarget cell.

BRIEF DESCRIPTION OF DRAWINGS

[0017]FIG. 1 is a perspective of the cell separation device made inaccordance with the teachings of the present invention;

[0018]FIG. 2 is an exploded perspective view of FIG. 1;

[0019]FIG. 3 is a perspective view of an alternate embodiment of thecapillary annulus;

[0020]FIG. 4 is a perspective view of another exemplary embodiment ofthe cell separation device made in accordance with the teachings of thepresent invention;

[0021]FIG. 5 is a perspective view of yet another exemplary embodimentof the cell separation device made in accordance with the teachings ofthe present invention;

[0022]FIG. 6 is an exploded perspective view of FIG. 4;

[0023]FIG. 7 is a perspective view of another exemplary embodiment ofthe cell separation device made in accordance with the teachings of thepresent invention; and

[0024]FIG. 8 is a top view of the flow chamber of FIG. 7.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to devices and methods of isolatingand identifying a desired target cell on a single substrate. Morespecifically, the desired target cell is isolated from the various cellsby targeting the various antigens found on the cell membrane of thetarget cell. That is, the present invention utilizes monoclonalantibodies that are directly conjugated to biotin and to a markermolecule wherein the conjugated antibodies are designed to bind toparticular antigens found on the target cells. The present inventionalso utilizes the unique binding affinity of biotin (or biotinderivatives) to avidin or streptavidin to isolate the desired targetcell from the cellular mixture. The target cells are separated from thecell suspension when the cell suspension is passed over an immobilizedavidin or streptavidin substrate. As a result, the conjugated cellsadhere to the avidin/streptavidin substrate, while the unbound cells arewashed off and collected in a wicking member. The captured cells canthen be reacted with other antibodies for subsequent detection andenumeration. Furthermore, the methods of the present invention allowsfor release of the target cells by chemical and/or mechanical methods.

[0026] The device and methods are advantageous over the prior art cellseparation devices. First, the present invention optimizes target cellrecovery and purity as the captured target cell may be easily identifiedand separated from the substrate. Rather, unlike prior art devices thatutilize spherical beads, the present invention utilizes a planarsubstrate where the target cell may be separated from a cell mixture andmay also be identified on the substrate. Consequently, the isolatedcells may easily be identified on a planar surface as compared to aspherical bead. Moreover, the present invention also eliminates thesteps of removing the target cell from the substrate for subsequentidentification. Rapid identification and capture of a specific targetcell is particularly important where the target cell, such asmicrometastases, has a very low frequency. Nevertheless, the devices andmethods of the present invention are also useful for identifying andcapturing target cells that have a lower or greater frequency thanmicrometastases.

[0027] Moreover, the target cells captured with the cell separationdevice of the present invention are uncompromised physically orbiochemically. Accordingly, the captured cells may be more easilyrecovered and analyzed as compared to prior art techniques. Unlike theprior art techniques, the isolated target cells of the present inventionare not directly bound to the substrate. Rather, the isolated targetcell is separated from a cell mixture by utilizing the binding affinityof avidin and biotin. Consequently, the isolated target cells are moreeasily separated from the substrate than is possible with target cellremoval from spherical beads or magnetic particles. Additionally, thedevice of the present invention is more efficient as compared to priorart devices and limits operator error. That is, according to the presentinvention, the target cell may be isolated, identified, and subsequentlyreleased from the same fixed substrate.

[0028] According to an exemplary embodiment of the present invention,the cell separation apparatus 10 comprises a fixed substrate 16 such as,but not limited to, a standard glass microscope slide. The fixedsubstrate 16 may be made from materials such as, but not limited to,metal, glass, plastic, or ceramic materials. As shown in FIG. 2, thefixed substrate 16 is provided with a bioactive coating 18 applied to adefined area that may have a plurality of shapes such as, but notlimited to, a circle, an oval, a square, or a rectangle. According toexemplary embodiments of the present invention, the bioactive coating 18may be avidin, streptavidin, or derivatives thereof. For purposes ofexample, but not of limitation, the bioactive coatings will be referredto as “avidin” coating.

[0029] The avidin coating 18 may be applied to the fixed substrate 16 bya number of methods. One method includes directly bonding avidin 18 tothe fixed substrate's surface. By directly attaching the avidin to thefixed substrate 16, covalent chemical bonding techniques are required.Generally, the fixed substrate 16 must possess chemical functionalgroups on its surface such as carbonyl groups, primary amines, hydroxylgroups, silane groups which will form a strong, chemical bond withsimilar groups on the active compound. In the absence of such chemicalforming functional group, techniques may be utilized to activate thematerial's surface before coupling the biological compound. Surfaceactivation is a process of generating, or producing, reactive chemicalfunctional groups using chemical or physical techniques such as, but notlimited to, ionization, heating, photochemical activation, oxidizingacids, and etching with strong organic solvents.

[0030] According to an alternative method, avidin 18 is indirectly boundto the fixed substrate's surface 16 through an intermediate layer (notshown). This intermediate layer may be either covalently bound to thefixed substrate's surface or bonded through strong intermolecularattractions such as ionic or Van der Waals forces. Examples of commonlyused intermediate layers include, but are not limited to, organicpolymers such as silicones, polyamines, polystyrene, polyurethane,acrylates, and methoxysilanes.

[0031] As shown in FIG. 2, the capillary annulus 20 is a generallycylindrical structure having a conduit 22 that extends along thelongitudinal axis of the cylindrical structure through which the cellsample may travel. The capillary annulus 20 may be made from a number ofdifferent materials such as, but not limited to, plastic, metal, orceramic. In an alternate embodiment, the capillary annulus 20 mayinclude a plurality of conduits that extend along the longitudinal axis.As shown in FIG. 1, the conduit 22 has a constant diameter along thelength of the conduit. In an alternate embodiment, the conduit 28 may betapered (conical) or have a decreasing radius as illustrated in FIG. 3.

[0032] The capillary annulus 20 is placed in spaced communication withthe fixed substrate 16. The distance between the base of the annulus andthe fixed substrate 16 is a factor in defining the flow rate of the cellsample across the avidin coated portion 18 of the fixed substrate 16.This distance is also related to the binding efficiency between thebiotinlyated antibodies and the avidin as the binding efficiency ispredicated on the contact time between the biotinlyated antibodies andthe avidin coating. In a preferred embodiment, this distance is in therange of 2 to 20 microns.

[0033] Optionally, the capillary annulus 20 may be provided with atleast one protuberance (not shown) extending axially from the edge ofthe base of the annulus. The protuberance(s) may be positioned along theperimeter of the annulus and is sized so that the annulus 20 isconsistently spaced a specified distance from the avidin coated area 18of the fixed substrate. As those skilled in the art will appreciate, theprotuberances may be sized differently to effect different distancesbetween the base of the capillary annulus 20 and the surface of thefixed substrate 16. Furthermore, the capillary annulus 20 may include atleast one fastening member such as, but not limited to, braces, latches,clamps, or hooks (not shown) provided about the periphery of theannulus. The fastening members serve to secure the annulus 20 to thefixed substrate 16. The fastening members are shaped to permit theattachment and removal of the annulus 20 from the substrate 16.

[0034] The cell separation device 10 may also include a specimen chamber12 that is in communication with the capillary annulus 20. The specimenchamber 12 is a generally cylindrical chamber having at least oneopening 24 provided on the bottom surface of the chamber thatcorresponds to the conduit opening 22 on the capillary annulus 20. In apreferred embodiment, the specimen chamber 12 is adapted to holdapproximately 5.0 mL to 25.0 mL of solution. The specimen chamber 12 maybe made from materials such as plastics, metals, or ceramics. Asillustrated in FIGS. 1-2, the specimen chamber 12 may be reversiblyattachable and detachable from the capillary annulus 20. In an alternateembodiment, those skilled in the art will appreciate that the capillaryannulus 20 and the specimen chamber 12 may be made from a single pieceof material.

[0035] As shown in FIGS. 1-2, a fluid absorbing media 14 surrounds thecapillary annulus 20. According to one embodiment, the fluid absorbingmedia 14 is reversibly attached to the fixed substrate 16 by a fasteningmeans (not shown) such as, but not limited to, braces, latches, clamps,or hooks. According to another embodiment of the present invention, thefluid absorbing media 14 may be permanently affixed to the fixedsubstrate 16 with glue or other bonding agents. The fluid absorbingmedia 14 may be comprise absorbent materials such as, but not limitedto, cellulose acetate, polyester, nylon, polyolefin, or blends thereof.According to one exemplary embodiment, the fluid absorbing media 14comprises thermal bonded extra absorbent materials supplied by FiltronaRichmond Inc. The fluid absorbing media 14 is sized to have sufficientcapacity to absorb at least a volume of cells equivalent to the maximumcapacity of the specimen chamber 12. Those skilled in the art willappreciate that the size and shape of the fluid absorbing media 14 maydeviate from what is depicted in FIG. 1.

[0036] The fluid absorbing media 14 absorbs the unbound cells andsolution that have been exposed to avidin 18 on the fixed substrate 16.Additionally, the absorptive properties of the media 14 also contributeto the binding efficiency between the biotinylated antibodies and theavidin coating. That is, the greater the absorptive properties of themedia 14, the less resulting contact time between the solution and theavidin 18. Accordingly, the absorptive efficiency of the fluid absorbingmedia 14 must be selected so as to optimize the time for thebiotinylated antibodies to bind to the avidin coating on the substrate16.

[0037] The cell separation device of the present invention also includesat least two types of conjugated antibodies. The antibodies may beconjugated with either a biotin molecule or a marker molecule. Moreover,the antibodies are specific for the target cells and are non-reactivewith the mixed cell population such as, but not limited to, blood, bonemarrow, or effusion fluids. For instance, according to one embodiment ofthe present invention, a pan-epithelial cell antibody may be utilized.Such antibody is directed to antigens present on epithelial cellmembranes. Accordingly, the cell separation device utilizing theseantibodies will target and separate those cells that have antigens thatreact with the pan-epithelial antibody. In an alternate embodiment,antibodies that are specific to certain blood cell antigens (known as CDantigens) may be utilized. For example, CD19 and CD20 antibodies may beused to selectively capture B lymphocytes in a mixed population of bloodor bone marrow cells. In yet another embodiment, antibodies toinfectious agents such as, but not limited to, cytomegalovirus or HIVmay also be used to selectively capture infectious cells in blood orbody fluid samples.

[0038] Additionally, the present invention includes antibodies that areconjugated with marker molecules such as, but not limited to,flurochromes, radiolabels, fluorescent agents, or chromophores. Theantibodies that are conjugated with a marker molecule allow the isolatedtarget cell to be easily identified on the avidin coated slide.Accordingly, the present invention provides a device wherein cellseparation and identification may occur on the same substrate.

[0039] Furthermore, according to alternate embodiments of the presentinvention, binder molecules, other than an antibodies, may be conjugatedwith biotin. The binder molecules include, but are not limited to,glycoconjugates, lectins, hormones, cell receptors, vitamins, aminoacids, sugars, lipids, fatty acids, liposomes, DNA probes, or RNAprobes. These binder molecules may be utilized to target particularlectins, enzymes, receptors, transport proteins, hydrophobic sites,membranes, nucleic acids, or genes.

[0040] FIGS. 4-6 illustrate other exemplary embodiments of the presentinvention. The cell separation device 30 comprises a fixed substrate 16having an avidin coating 18 provided thereon, a specimen chamber 34, acapillary flow chamber 32 and a fluid absorbing media 36. Like theprevious embodiment of the present invention, the avidin is provided ona defined area of the fixed substrate 16. The capillary flow chamber 32is in communication with the fixed substrate 16 and spans at least theavidin coated region 18 of the fixed substrate 16. The capillary chamber32 is a housing having a top wall, a first side wall, and a second sidewall. The top wall is configured such that the top wall is approximatelyparallel to the surface of the fixed substrate 16. The first and secondside walls span between the top wall and the surface of the fixedsubstrate 16 to define a conduit. The side walls are configured suchthat the capillary flow chamber 32 is reversibly attached to the fixedsubstrate 16 by a fastening means (not shown). The fastening means maybe fasteners such as, but not limited to, braces, latches, clamps, orhooks. According to another embodiment, the capillary flow chamber 32may be permanently affixed to the fixed substrate 16.

[0041] Generally, the specimen chamber 34 is provided at the first endof the capillary flow chamber 32 and a fluid absorbing media 36 isprovided at the second end of the capillary flow chamber 32. Forinstance, FIG. 5 illustrates one embodiment wherein the fluid absorbingmedia 36 is external to the capillary flow chamber 32, and FIG. 6illustrates a second embodiment wherein the fluid absorbing media 36 ispositioned within the capillary flow chamber 32. With respect to thesecond embodiment as depicted in FIG. 6, the fluid absorbing media 36 ispositioned downstream of the avidin coating.

[0042] FIGS. 7-8 illustrate another exemplary embodiment of the presentinvention. The cell separation device 40 comprises a fixed substrate 42having an avidin coating 44 provided thereon, a flow chamber 46, and afluid absorbing media 48. Like previous embodiments, the avidin isprovided on defined area of the fixed substrate 42. According to oneembodiment, the avidin coating comprises a surface area of approximately8 cm². Those skilled in the art will appreciate that the surface areamay vary in size and is also dependent on the size of the fixedsubstrate 42.

[0043] As shown in FIG. 8, the flow chamber 46 is a housing capable ofholding a fluid. According to one exemplary embodiment, the flow chamber46 may be sized to contain approximately 24 mL of fluids. However, thoseskilled in the art will appreciate that the capacity of the flow chambermay be varied by altering the size of the flow chamber or the height ofthe walls. The base of the housing also includes a plurality of holes 50that allow the cell sample to be delivered onto the avidin coatedportion 44 of the fixed substrate 42. Those skilled in the art willappreciate that the size, geometry, and density of holes on the housingbase may be varied to alter or optimize fluid flow rates onto the fixedsubstrate which will also effect target cell capture characteristics ofthe fixed substrate.

[0044] The flow chamber 46 also includes at least one fastening means 52to attach and detach the flow chamber to the fixed substrate 42. Thefastening means may be fasteners such as, but not limited to, braces,latches, clamps, or hooks. According to one embodiment, the fasteningmeans is positioned on the fixed substrate so as to provide clearancedistance between the base of the flow chamber and the fixed substrate.Accordingly, like other embodiments of the present invention, thisdistance is a factor in regulating the flow rate of the cell sampleacross the avidin coated portion 44 of the fixed substrate 42.Alternatively, the flow chamber 46 may be directly affixed to thesurface of the fixed substrate 42. According to this exemplaryembodiment, the flow chamber 46 is provided with a skirt (not shown)around the base of the flow chamber. The skirt is also provided with ordefines at least one slot (not shown). The slot allows fluid to travelfrom the avidin portion of the fixed substrate 42 to the fluid absorbingmedia 48. According to one exemplary embodiment, the flow chamber 46 isprovided with one slot that extends along the main axis of the flowchamber as shown in FIG. 7. According to another exemplary embodiment,slots may be positioned on opposing sides of the flow chamber.

[0045] As shown in FIG. 7, fluid absorbing media 48 is positioned alongone edge of the fixed substrate. However, in alternate embodiments,those skilled in the art will appreciate that fluid absorbing media maybe positioned on one or more edges of the fixed substrate to absorb thecell sample solution or washing fluids that may accumulate on thesurface of the fixed substrate. The fluid absorbing media 48 is sized sothat it may accommodate up to approximately 60 mL of fluid.

[0046] In operation, a cell sample is loaded into the specimen chamber.The cell sample flows across the slide, and those cells bound tobiotinlyated antibodies are removed from the cell sample as the biotinreacts with the avidin. The binding between the biotin and the avidin isadjustable by varying the flow rate of the cell sample across the avidincoated portion of the fixed substrate. The flow rate may be increased bypositioning the capillary flow chamber such that the specimen chamber isabove the fluid absorbing media. For instance, the fixed substrate isangled such that the cell sample flows downstream towards the fluidabsorbing media. Alternately, the absorptive properties of the fluidabsorbing media may be increased or decreased which may increase ordecrease the flow of cell sample over the avidin coating. Furthermore, acombination of the absorptive properties of the fluid absorbing mediaand the incline of the slide may also be adjusted to varying the flowrate of the cell sample across the avidin coated portion of the fixedsubstrate.

[0047] The present invention also relates to methods of isolatingspecific cells from a mixture of different cells. According to onemethod of the present invention, the cell preparation is de-bulked ofred blood cells using standard laboratory procedures. The preparation isthen incubated with a mixture of biotinylated antibodies [mAb_(b)] andfluorescent-conjugated antibodies [mAb_(f)]. The mAb_(b) and the mAb_(f)components bind to the target cells in approximate equal proportion. Thecell preparation is subsequently washed to remove unbound antibodies andloaded into the specimen-loading chamber of the device. In oneembodiment, the loading chamber is designed to accommodate liquidvolumes ranging from approximately 5.0 ml to 25.0 ml. The cellpreparation then flows through the capillary annulus at a preset flowrate that allows the mAb_(b) on the target cell surface to bind to theavidin substrate on the slide. Cells that do not bind (non-target cells)are pulled into the absorbent wicking pad by passive absorption.

[0048] Washing fluids such as, but not limited to, phosphate bufferedsaline or water may be then applied into the specimen-loading chamber ata controlled flow rate. This provides for additional movement of unboundcells into the wicking pad. The specimen loading chamber, capillaryannulus, and wicking pad may be then removed and the collected targetcells may be viewed under a fluorescent microscope. Optionally, theslide may then be fixed with an aldehyde or cell-preservation media forpermanence.

[0049] According to another exemplary method of the present invention,the cell isolating device may be used to obtain viable target cells.First, the targeted cell is isolated by the preceding methods. After thespecimen loading chamber, capillary annulus, and wicking pad are removedfrom the slide, a second specimen loading chamber and capillary annulus(without a wicking pad) are attached to the slide. Cell releasing agentsare loaded into the specimen-loading chamber, and they are allowed tocontact the slide. Following a brief incubation time, the chamber isthen loaded with a solution (e.g., isotonic buffer, protein medium insolution) that stops the cell releasing chemical reaction. The releasedtarget cells are in solution and may be collected in a secondarycollection vessel. According to an alternate embodiment, a port may beprovided on the annulus/slide interface to allow for the collection ofthe solution containing the target cells. The cells contained within thesecondary collection vessel then may be reacted with other antibodiesfor subsequent detection and enumeration. According to an alternatemethod of the present invention, the released cells may be subsequentlyanalyzed by molecular biology techniques for particular cellular genesand/or proteins.

[0050] According to one embodiment of the present invention, the cellreleasing agent may be a low pH buffer solution having a pH ranging fromabout 3.0 to 6.0. Examples of the buffer solution that may be adjustedto the desired pH range include, but are not limited to, TRIS(Tris(hydroxymethyl)aminomethane) buffer, phosphate-buffered saline, andlithium carbonate. According to another embodiment, the cell releasingagent may be enzymes such as, but not limited to, chymopapain, trypsin,chymotrypsin, and V8 protease. According to yet another embodiment, thecell releasing agent may be a chemical reagent that cleaves peptide suchas, but not limited to, EDTA (Ethylenediaminetetraacetic Acid), cyanogenbromide, or 2-Nitro-5-thiocyanobenzoate.

[0051] According to yet another exemplary method of the presentinvention, viable target cells may be separated from the cell isolatingdevice. First, the targeted cell is isolated by the preceding methods.After the specimen loading chamber, capillary annulus, and wicking padare removed from the slide, a second specimen loading chamber andcapillary annulus (without a wicking pad) are attached to the slide. Thesecond specimen loading chamber is then loaded with an isotonic buffersolution. The isotonic buffer is then allowed to contact the cellisolating device. The buffer solution on the cell isolating device isthen mechanically agitated thereby resulting in the breaking of theantigen-antibody bond. As a result, the target cell may be released intothe buffer solution and collected for subsequent analysis. According toone exemplary method, the buffer solution may be agitated by sonicationor other means of shaking a solution known to those skilled in the art.According to another exemplary method, the cell isolating device may bephysically manipulated by sonication or other means to cause the targetcells to be separated from the cell isolating device.

[0052] In closing, it is to be understood that the embodiments andexamples of the present invention are illustrative of the principles ofthe present invention. Other modifications that may be employed arewithin the scope of the present invention; thus, by way of example, butnot of limitation, alternative configurations and methods of the presentinvention are also contemplated. Accordingly, the present invention isnot limited to that precisely shown and described herein.

What is claimed:
 1. A device for recovering a target cell from acellular matrix, comprising: a housing having at least one wall and abase, said base having a plurality of openings; a reaction surfacepositioned proximate to said plurality of openings; and a fluidabsorbing media in communication with said substrate and adjacent tosaid housing.
 2. The device of claim 1 wherein said housing furtherincludes at least one fastening member capable of engaging said reactionsurface.
 3. The device of claim 1 wherein said housing and said reactionsurface are in spaced relationship.
 4. The device of claim 3 whereinsaid reaction surface and said housing are separated by a distance ofapproximately 2 to 20 microns.
 5. The device of claim 1 wherein saidreaction surface comprises a bioactive coating selected from the groupconsisting of avidin, streptavidin, avidin derivatives, streptavidinderivatives, and blends thereof.
 6. The device of claim 5 wherein saidbioactive coating has a surface area of approximately 8 cm².
 7. A methodof recovering a target cell from a cellular mixture comprising: exposinga cellular mixture to a targeting solution to form a target cellmixture; delivering said target cell mixture to a reaction surface;controlling the delivery of said target cell mixture so as to optimizethe flow over said reaction surface; retaining said target cells on saidreaction surface; delivering a releasing solution to said reactionsurface; and collecting said releasing solution, wherein said releasingsolution includes said target cells.
 8. A method as set forth in claim7, wherein the controlling of the delivery of the target cell mixture isperformed by establishing a predetermined delivery distance for saidtarget cell solution to traverse to said reaction surface.
 9. A methodas set forth in claim 8 wherein the predetermined delivery distance isapproximately 2 to 20 microns.
 10. A method as set forth in claim 8,wherein the controlling the delivery of the target cell mixture furtherincludes absorbing non-reacting amounts of said target cell solutionfrom said reaction surface at a predetermined rate.
 11. The method ofclaim 10 further comprising delivering another aliquot of the targetcell releasing solution to said reaction surface, mechanically agitatingsaid target cell releasing solution, and collecting said target cellreleasing solution, wherein said target cell releasing solution includessaid target cells.
 12. The method of claim 7 wherein said targetingsolution includes a marker compound comprising a binder molecule and aprobe.
 13. The method of claim 12 wherein said binder molecule isselected from the group consisting of antibodies, antigens,glycoproteins, lectins, hormones, cell receptors, vitamins, amino acids,sugars, lipids, fatty acids, liposomes, DNA probes, and RNA probes. 14.The method of claim 13 wherein said probe is selected from the groupconsisting of flurochromes, radiolabels, fluorescent agents, andchromophores.
 15. The method of claim 7 wherein said reaction surfacecomprises a bioactive coating selected from the group consisting ofavidin, streptavidin, avidin derivatives, streptavidin derivatives, andblends thereof.
 16. The method of claim 7 wherein said releasingsolution is a buffer solution having a pH ranging from about 3.0 to 6.0.17. The method of claim 7 wherein said releasing solution includes acell releasing agent selected from the group consisting of chymopapain,trypsin, chymotrypsin, V8 protease, Ethylenediaminetetraacetic Acid,cyanogen bromide, or 2-Nitro-5-thiocyanobenzoate.
 18. A method ofisolating target cells from a cellular mixture comprising: exposing acellular mixture to a targeting solution to form a target cell mixture;delivering said target cell mixture to a reaction surface; controllingthe delivery of said target cell mixture so as to optimize the flow oversaid reaction surface; retaining said target cells on said reactionsurface; analyzing said target cells remaining on said reaction surface;and delivering at least one target cell releasing solution to saidreaction surface, and collecting said target cell releasing solution,wherein said target cell releasing solution includes said target cells.19. A method as set forth in claim 18, wherein the controlling of thedelivery of the target cell mixture is performed by establishing apredetermined delivery distance for said target cell solution totraverse to said reaction surface.
 20. A method as set forth in claim 19wherein the predetermined delivery distance is approximately 2 to 20microns.
 21. A method as set forth in claim 19, wherein the controllingthe delivery of the target cell mixture further includes absorbingnon-reacting amounts of said target cell solution from said reactionsurface at a predetermined rate.
 22. A method as set forth in claim 21further comprising delivering another aliquot of the target cellreleasing solution to said reaction surface, mechanically agitating saidtarget cell releasing solution, and collecting said target cellreleasing solution, wherein said target cell releasing solution includessaid target cells.
 23. The method of claim 18 wherein said targetingsolution includes a marker compound comprising a binder molecule and aprobe.
 24. The method of claim 23 wherein said binder molecule isselected from the group consisting of antibodies, antigens,glycoproteins, lectins, hormones, cell receptors, vitamins, amino acids,sugars, lipids, fatty acids, liposomes, DNA probes, and RNA probes. 25.The method of claim 24 wherein said probe is selected from the groupconsisting of flurochromes, radiolabels, fluorescent agents, andchromophores.
 26. The method of claim 18 wherein said reaction surfacecomprises a bioactive coating selected from the group consisting ofavidin, streptavidin, avidin derivatives, streptavidin derivatives, andblends thereof.
 27. The method of claim 18 wherein said target cellreleasing solution is a buffer solution having a pH ranging from about3.0 to 6.0.
 28. The method of claim 18 wherein said releasing solutionincludes a cell releasing agent selected from the group consisting ofchymopapain, trypsin, chymotrypsin, V8 protease,Ethylenediaminetetraacetic Acid, cyanogen bromide, or2-Nitro-5-thiocyanobenzoate.
 29. A method of recovering viable targetcells from a cellular mixture, comprising: providing an apparatus ofclaim 1; exposing the cellular mixture to a targeting solution to form atarget cell mixture; adding the target cell mixture to the apparatus;controlling the rate of contact between the reaction surface and thetarget cell mixture by establishing a predetermined delivery distancefor said target cell mixture to traverse to the reaction surface;analyzing the reaction surface for target cells; and removing targetcells from the reaction surface by delivering a releasing solution tothe reaction surface; and collecting the releasing solution, wherein thereleasing solution includes the target cells.